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AAFS 2011 ­ J.M. Butler ­ Additional STR Loci

February 24, 2011

American Academy of Forensic Sciences

February 24, 2011 ­ Chicago, IL

NIST Human Identity Project Teams

within the Applied Genetics Group

Forensic DNA Team

Data Analysis Support

Impact of Additional STR Loci on Random Match Probability Calculations and Kinship Analysis

John M. Butler,

Carolyn R. Hill, David L. Duewer, Kristen L. O'Connor

National Institute of Standards and Technology Gaithersburg, Maryland

John Butler

DNA Biometrics Team

Mike Coble

Becky Hill

Margaret Kline

Dave Duewer

Pete Vallone

Erica Butts

Kristen Lewis O'Connor

Funding from the National Institute of Justice (NIJ) through NIST Office of Law Enforcement Standards

Funding from the FBI S&T Branch through NIST Information Access Division

NIST and NIJ Disclaimer

Funding: Interagency Agreement between the National Institute of Justice and NIST Office of Law Enforcement Standards

Presentation Outline

· Need for additional loci

­ Growth in U.S. and other national DNA databases

Points of view are mine and do not necessarily represent

the official position or policies of the US Department of Justice or the National Institute of Standards and Technology. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.

· · · · ·

New STR kits available with additional loci Locus characteristics Population data and statistical analysis Random match probabilities (unrelated) Relationship likelihood ratios (related)

AAFS 2009 CRIMINALISTICS Session I:

Slide from my 2009 AAFS talk...

Envisioning the Future

Additional STR Loci in the Future?

· Will be needed for more complex kinship analyses and extended applications

­ Example: Y-STRs needed for familial searching

Forensic DNA:

Perspectives on Progress in a Rapidly Growing Field

John M. Butler, PhD

National Institute of Standards and Technology AAFS Meeting (Denver, CO) February 19, 2009

· Immigration testing needs more than 13 STRs · Larger DNA databases will require more loci

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

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AAFS 2011 ­ J.M. Butler ­ Additional STR Loci

February 24, 2011

Growth in Numbers of DNA Profiles Present in Various NDIS Indices

(cumulative totals by year)

Year ending Dec 31

Slide from my 2009 AAFS talk...

Possible scenarios for extending sets of genetic markers to be used in national DNA databases

Core set of markers

(e.g., CODIS 13 STRs)

Forensic

Convicted Offender

Arrestee

Total Offender*

Past and Present

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

21,625 441,181 -441,181 27,897 750,929 -750,929 46,177 1,247,163 -1,247,163 70,931 1,493,536 -1,493,536 In the last2,038,514 two years (2009, 2010): 93,956 -2,038,514 126,315 2,826,505 -2,826,505 103,008 forensic samples added 160,582 3,977,433 54,313 2,693,6355,287,505 offender 85,072 samples4,031,748 added 203,401 5,372,773 248,943 6,398,874 140,719 6,539,919 298,369 7,389,917 351,926 7,743,329 8,559,841 668,849 351,951 9,233,554

Source: FBI Laboratory's CODIS Unit

(a)

Extra loci would be included (due to large PCR multiplexes)

(b) Future (c) (d) Some loci may be dropped to enable replacement with better loci

Highly unlikely to start over with new loci

Position of Forensic STR Markers on Human Chromosomes

TPOX D3S1358 TH01 D5S818 FGA CSF1PO D8S1179 D7S820 VWA

23 Autosomal STR Markers

Present in Commercial STR Multiplex Kits

U.S. TPOX CSF1PO D5S818 D7S820 D13S317 FGA vWA D3S1358 D8S1179 D18S51 D21S11 TH01 D16S539 D2S1338 D19S433 Penta D Penta E Europe

13 Core U.S. STR Loci

1997 13 CODIS loci

ESS = European Standard Set

FGA vWA D3S1358 D8S1179 D18S51 D21S11 TH01 D16S539 D2S1338 D19S433

7 ESS loci

8 STR loci overlap between U.S. and Europe

AMEL

Sex-typing

D13S317 D16S539 D18S51 D21S11 AMEL

3 miniSTR loci developed at NIST

D12S391 D1S1656 D2S441 D10S1248 D22S1045 SE33

5 loci adopted in 2009 to expand to 12 ESS loci

Commercially Available STR Kits

Applied Biosystems (17) · AmpFlSTR Blue (1996) · AmpFlSTR Green I (1997) · Profiler (1997) · Profiler Plus (1997) · COfiler (1998) · SGM Plus (1999) · Identifiler (2001) · Profiler Plus ID (2001) · SEfiler (2002) · Yfiler (2004) · MiniFiler (2007) · SEfiler Plus (2007) · Sinofiler (2008) ­ China only · Identifiler Direct (2009) · NGM (2009) · Identifiler Plus (2010) · NGM SElect (2010) Promega Corporation (13) · PowerPlex 1.1 (1997) · PowerPlex 1.2 (1998) · PowerPlex 2.1 (1999) · PowerPlex 16 (2000) · PowerPlex ES (2002) · PowerPlex Y (2003) · PowerPlex S5 (2007) · PowerPlex 16 HS (2009) · PowerPlex ESX 16 (2009) · PowerPlex ESX 17 (2009) · PowerPlex ESI 16 (2009) · PowerPlex ESI 17 (2009) · PowerPlex 18D (2011) Qiagen (10) kits in 2010

Primarily selling kits in Europe Due to patent restrictions cannot sell in U.S.

Same DNA Sample Tested with Five STR Kits

Identifiler (Applied Biosystems)

Investigator kits · ESSplex · ESSplex SE · Decaplex SE · IDplex · Nonaplex ESS · Hexaplex ESS · HDplex · Triplex AFS QS · Triplex DSF · Argus X-12

NGM SElect (Applied Biosystems)

PowerPlex 16 (Promega)

PowerPlex ESX 17 (Promega)

~1/3 of all STR kits were released in the last two years

ESSplex (Qiagen)

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

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AAFS 2011 ­ J.M. Butler ­ Additional STR Loci

February 24, 2011

J.M. Butler (2011), Advanced Topics in Forensic DNA Typing: Methodology , Figure 5.5

13 CODIS STR Loci

Additional 10 STRs

STR Kit Concordance Testing

· Many of these STR kits have different primer sequences for amplifying the same STR locus · Need to analyze the same DNA samples with different STR typing kits looking for differences · In some rare cases, allele dropout may occur due to mutations in primer binding regions

See Becky Hill's talk on Friday morning at 10 a.m. ( Criminalistics, A110) "Concordance Testing Comparing STR Multiplex Kits with a Standard Data Set"

The 10 STR Loci Beyond the CODIS 13

STR Locus D2S1338 D19S433 Penta D Penta E

5 new European loci

31 STR Typing Kits 25 Alleles Reported in the Literature for D1S1656

Allele

(Repeat #)

Location 2q35 19q12 21q22.3 15q26.2 1q42 12p13.2 2p14 10q26.3 22q12.3 6q14

Repeat Motif

TGCC/TTCC AAGG/TAGG AAAGA AAAGA TAGA AGAT/AGAC TCTA/TCAA GGAA ATT

5.2 to 20 1.1 to 19 5 to 32 8 to 20.3 13 to 27.2 8 to 17 7 to 19 7 to 20 3 to 49

36 50 53 25 52 22 13 14 178

D1S1656 D12S391 D2S441 D10S1248 D22S1045 SE33

AAAG

*Allele range and number of observed alleles from Appendix 1, J.M. Butler (2011) Advanced Topics in Forensic DNA Typing: Methodology; SE33 alleles have complex repeat structure

15 NIST observed alleles circled in red

Allele Range* 10 to 31

# Alleles* 40

8 9 10 (a) 10 (b) 11 12 (a) 12 (b) 13 (a) 13 (b) 13.3 14 (a) 14 (b) 14.3 15 15.3 16 16.3 17 17.1 17.3 18 18.3 19 19.3 20.3

Promega ESX 17 133 bp 137 bp 141 bp 141 bp 145 bp 149 bp 149 bp 153 bp 153 bp 156 bp 157 bp 157 bp 160 bp 161 bp 164 bp 165 bp 168 bp 169 bp 170 bp 172 bp 173 bp 176 bp 177 bp 180 bp 184 bp

Promega ESI 17 222 bp 226 bp 230 bp 230 bp 234 bp 238 bp 238 bp 242 bp 242 bp 245 bp 246 bp 246 bp 249 bp 250 bp 253 bp 254 bp 257 bp 258 bp 259 bp 261 bp 262 bp 265 bp 266 bp 269 bp 273 bp

ABI NGM 171 bp 175 bp 179 bp 179 bp 183 bp 187 bp 187 bp 191 bp 191 bp 194 bp 195 bp 195 bp 198 bp 199 bp 202 bp 203 bp 206 bp 207 bp 208 bp 210 bp 211 bp 214 bp 215 bp 218 bp 222 bp

Repeat Structure Reference [TAGA]4[TGA]0-1[TAGA]nTAGG[TG]5 [TAGA]8[TG]5 Phillips et al. (2010) [TAGA]9[TG]5 Phillips et al. (2010) [TAGA]10[TG]5 Lareu et al. (1998) [TAGA]10TAGG[TG]5 Phillips et al. (2010) [TAGA]11[TG]5 Lareu et al. (1998) [TAGA]12[TG]5 Lareu et al. (1998) [TAGA]11TAGG[TG]5 Lareu et al. (1998) [TAGA]12TAGG[TG]5 Lareu et al. (1998) [TAGA]13[TG]5 Phillips et al. (2010) [TAGA]1TGA[TAGA]11TAGG[TG]5 Phillips et al. (2010) [TAGA]13TAGG[TG]5 Lareu et al. (1998) [TAGA]14[TG]5 Phillips et al. (2010) [TAGA]4TGA[TAGA]9TAGG[TG]5 Phillips et al. (2010) [TAGA]14TAGG[TG]5 Lareu et al. (1998) [TAGA]4TGA[TAGA]10TAGG[TG]5 Lareu et al. (1998) [TAGA]15TAGG[TG]5 Lareu et al. (1998) [TAGA]4TGA[TAGA]11TAGG[TG]5 Lareu et al. (1998) [TAGA]16TAGG[TG]5 Lareu et al. (1998) Not published Schröer et al. (2000) [TAGA]4TGA[TAGA]12TAGG[TG]5 Lareu et al. (1998) [TAGA]17TAGG[TG]5 Phillips et al. (2010) [TAGA]4TGA[TAGA]13TAGG[TG]5 Lareu et al. (1998) Not published Asamura et al. (2008) [TAGA]4TGA[TAGA]14TAGG[TG]5 Lareu et al. (1998) Not published Gamero et al. (2000)

from Appendix 1, J.M. Butler (2011) Advanced Topics in Forensic DNA Typing: Methodology

NIST U.S. Population Allele Frequencies

D1S1656 (15 different alleles)

African American Allele (N = 341) 10 0.01433 11 0.04871 12 0.06304 13 0.10029 14 0.25788 14.3 0.00716 15 0.15616 15.3 0.03009 16 0.11032 16.3 0.10029 17 0.02865 17.3 0.05014 18 0.00287 18.3 0.02436 19.3 0.00573 Caucasian (N = 361) 0.00277 0.07756 0.11773 0.06648 0.07895 0.00277 0.14820 0.05817 0.13573 0.06094 0.04709 0.13296 0.00554 0.05125 0.01385 Hispanic (N = 236) 0.00630 0.02731 0.08824 0.11555 0.11765 0.00420 0.13866 0.05042 0.17437 0.05462 0.04202 0.14496 0.00630 0.02521 0.00420

D1S1656 Characteristics

· · · · 15 alleles observed 92 genotypes observed >89% heterozygotes (heterozygosity = 0.8934) 0.0220 Probability of Identity (PI)

N = 938

(only unrelated samples used; fathers removed from this sample set) < 5/2N

15 different alleles

PI = ( genotype frequencies ) 2

These values have been calculated for all 23 STR loci across the U.S. population samples examined

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

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AAFS 2011 ­ J.M. Butler ­ Additional STR Loci

February 24, 2011

Loci sorted on Probability of Identity (PI) values

Alleles Genotypes STR Locus Observed Observed SE33 53 292 Penta E* 20 114 D2S1338 13 68 D1S1656 15 92 D18S51 21 91 D12S391 23 110 FGA 26 93 Penta D* 16 71 D21S11 25 81 D19S433 16 76 D8S1179 11 45 vWA 11 38 D7S820 11 32 TH01 8 24 D16S539 9 28 D13S317 8 29 D10S1248 12 39 D2S441 14 41 D3S1358 11 30 D22S1045 11 42 CSF1PO 9 30 D5S818 9 34 TPOX 9 28 Het. (obs) 0.9360 0.8799 0.8785 0.8934 0.8689 0.8795 0.8742 0.8754 0.8358 0.8124 0.7878 0.8060 0.8070 0.7580 0.7825 0.7655 0.7825 0.7772 0.7569 0.7697 0.7537 0.7164 0.6983

PI value 0.0069 0.0177 0.0219 0.0220 0.0256 0.0257 0.0299 0.0356 0.0410 0.0561 0.0582 0.0622 0.0734 0.0784 0.0784 0.0812 0.0837 0.0855 0.0873 0.0933 0.1071 0.1192 0.1283

N = 938

23 STR Loci present in STR kits

rank ordered by their variability Better for mixtures (more alleles seen)

Random Match Probability for Various Combinations (assuming unrelated individuals)

STR Marker Combinations RMP* 6.0E-16 7.3E-19 4.9E-22 2.8E-25 1.2E-30 1 in ... 14 orders of magnitude improvement 1.7E+15 1.4E+18 2.0E+21 3.6E+24 8.4E+29

13 CODIS STRs 15 STRs

(+D2S1338, D19S433)

There are several loci more polymorphic than the current CODIS 13 STRs

18 STRs

(+D2S441, D10S1248, D22S1045)

20 STRs

(+D1S1656, D12S391)

Better for kinship (low mutation rate)

23 STRs

(+SE33, Penta D, Penta E)

*RMP values calculated by combining Probability of Identity values for each locus

More Loci are Useful

in Situations Involving Relatives

· Missing Persons and Disaster Victim Identification (kinship analysis) · Immigration Testing (often limited references)

­ Recommendations for 25 STR loci

In tomorrow's issue (February 25, 2011) of Forensic Science International...

· Deficient Parentage Testing

­ often needed if only one parent and child are tested

Disaster victim identification from the 2009 Victorian bushfires relied on DNA

Relationship testing labs are being pushed to answer more difficult genetic questions...and we want to make sure the right tools are in place

(82% involved kinship associations rather than direct matching) They advocate additional autosomal STR loci to aid kinship associations

How do 13 loci perform for kinship analysis?

Unrelated Related

Parent-Offspring

(1000 simulations)

Do additional loci improve kinship determination?

CODIS 13 15 STRs 18 STRs 20 STRs 23 STRs

Median values 10,000 77,000 830,000 5,700,000 240,000,000

median LR=2.7E-13

median LR=10,000

Proportion of simulations using U.S. Caucasian allele frequencies

Proportion of simulations using U.S. Caucasian allele frequencies

The degree of overlap corresponds with possible values for false positive or false negative results.

Parent-offspring

1000 simulations

Parent-offspring comparisons:

Full Siblings

(5000 simulations)

0.668 < -10

No overlap between unrelated and related LR distributions

median LR=1.5E-3

median LR=2400

Full sibling comparisons:

False positive rate = 0.027 False negative rate = 0.033

Median values 2,400 10,500 83,000 460,000 9,300,000

Full siblings

5000 simulations (additional simulations performed for smoother curves)

Half Siblings

(1000 simulations)

Half sibling comparisons:

False positive rate = 0.155 False negative rate = 0.168

median LR=0.14

median LR=6.9

Median values 7 12 20 32 82

Half siblings

1000 simulations

LR threshold = 1

log10(LR)

LR = 1

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

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AAFS 2011 ­ J.M. Butler ­ Additional STR Loci

February 24, 2011

Summary

· Additional autosomal STR loci exist in new STR kits and are being studied at NIST in U.S. population sample sets · To avoid potential adventitious matches with large DNA databases, enable greater international data sharing, and aid missing persons applications, it is highly likely that additional loci will be added to the U.S. core in the future

Thank you for your attention

Contact Information John Butler NIST Fellow Group Leader of Applied Genetics [email protected] 301-975-4049 http://www.cstl.nist.gov/biotech/strbase

Our team publications and presentations are available at: http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

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